As part of studies aimed at understanding mechanisms of membrane exchange between the cell surface and cytoplasmic membrane compartments, we have labeled the surface of the small soil amoeba, Acanthamoeba castellanii, with a gold probe, followed the internalization of the probe by electron microscopy, and measured the distribution of the probe by morphometric analysis of the micrographs. The probe, a monoclonal antibody coupled directly to colloidal gold, was specific to a subset of plasma membrane proteins. The probe was bound to the cell surface at 4 degrees C. and its internalization was followed over a period of 40 min. The label was internalized in endocytic vesicles ranging from >0.2 up to about 1.0 (mu)m in diameter. The gold label appeared in two major membrane compartments: intermediate vesicles, with diameters in the range of 0.2-0.7 (mu)m and vacuoles, larger than 0.8(mu)m in diameter. The latter have been previously identified as the site of hydrolytic enzymes and correspond to the amoeba's digestive organelles. Both these membrane compartments became labeled in an approximately linear fashion with a corresponding linear decrease in the unlabeled compartment. Their total volume fraction was essentially constant during the period of the experiment. There was morphological evidence of considerable fusion and/or fission in both compartments. Vesicles were labeled first in increasing numbers with time. A slight lag in labeling of vacuoles was evident. After 40 min there was evidence of removal of the probe from the membrane of the vacuole by digestion. The data suggest a sequential movement of membrane from the cell surface through a cytoplasmic vesicular compartment into the vacuolar digestive system. The movement of membrane appears to occur primarily by vesicular transport.